The nonmuscle myosin heavy chain is an ubiquitous protein present in all eukaryotic cells. Its real function is not very well know, but it is clear that its is involved not only in the motility of the cells, but also in the cellular cytokinesis and capping. This laboratory had isolated two different CDNAS in human and chicken tissues encoding for nonmuscle myosin heavy chains (NMMHCs). These two isoforms are called NMMHC-A and -B and are located on two different chromosomes. To understand the function of the two isoforms of the NMMHCs, we are trying to perform a knockout of one of the two genes using homologous recombination in embryonic stem cells and later in transgenic mice. The aim of the project is to see what happens when one of the two proteins encoded by the two isoforms is eliminated. To clone the NMMHC-A isoform, a mouse genomic library constructed in a cosmid vector was screened using a 5' portion CDNA human probe (HA-302, 1.2 kb). One positive clone is being characterized. For the NMMHC-B, a mouse genomic library constructed in lambda phage was screened using a 5' CDNA human probe (HB 5.12, 1.7 kb). In this case, 40 plaques appeared positive and, of these, one is being characterized. Human oligonucleotide probes were used to confirm if the 5' upstream sequence of the genes was present in our colonies. After that, fragments of about 5 kb containing the ATG sequence were subcloned in the Bluescript SBK+ plasmid to be used for homologous recombination. Restriction maps of these fragments from both genes were performed and the digestion products were hybridized with oligonucleotides containing the ATG sequence. These genomic mouse fragments will be used to introduce a neomycin sequence after the ATG site in order to disrupt the protein translation of NMMHCs. This construct will be introduced into embryonic stem cells and then in transgenic mice.